The present invention relates to a novel method for the preparation of nanoparticles formed from a polymerised methylidene malonate compound, said nanoparticles, optionally containing one or more biologically active molecules, as well as to pharmaceutical compositions containing them.
xe2x80x9cNanoparticlesxe2x80x9d is understood as meaning sub-micron particles having a diameter of less than about 500 nanometers. Nanoparticles formed by emulsion polymerisation of an alkyl cyanoacrylate are described in the EP 0 007 895 patent. The method used in the preparation of these alkyl cyanoacrylate particles relies on the (anionic) polymerisation of the monomer which takes place spontaneously and in an aqueous medium. The preparation which follows the same principle (anionic emulsion polymerisation) of nanoparticles constituted of a methylidene malonate polymer is described notably in F. Lescure et al, Pharm. Res., 1994, 11, 1270-1276. These monomers, whose preparation is described in the EP 0 283 364 patent, have a structure close to that of the cyanoacrylates but the nitrile function of the latter is replaced with an ester or an ester ester. Like the cyanoacrylates, they polymerise in the cold in an aqueous medium and can be biodegradable.
However, the methylidene malonate nanoparticles thus obtained possess certain drawbacks.
In fact, the emulsion polymerisation of methylidene malonates in the form of nanoparticles leads, in aqueous phase and at slightly acid pH, to the formation of oligomers, mainly of the trimer or tetramer type, which are highly biodegradable. These molecular species are partially hydrosoluble, such that the dispersion of these nanoparticles in an aqueous medium leads to their solubilisation and to the rapid loss of the particle structure (P. Breton et al., Eur. J. Pharm. Biopharm., 1996, 42, 95-103). When a biologically active molecule is associated with the methylidene malonate nanoparticles, is therefore possible for the molecule to be released very rapidly after the administration, following the effect of dilution in the circulatory current which brings about the rapid solubilisation of the oligomers which form the particle matrix, before eventually arriving at the site of action of the active principle.
Certain experiments have shown that the polymerization at basic pH enabled the formation of polymers of higher molecular masses while maintaining the size of the nanoparticles. However, such syntheses are characterised by:
the impossibility of obtaining polymers of Mw less than 10 000, and a fortiori Mw less than 8000. constituting individualised nanoparticles, without forming aggregates and without the significant presence of oligomeric species.
the impossibility of constituting polymers of Mw greater than 20 000 and a fortiori of higher Mw, at high pH (pH greater than 7) without the inevitable formation of aggregates which render the intravascular administration of these preparations impossible.
xe2x80x9cMwxe2x80x9d is understood as meaning the mass average molecular mass (or average molecular mass) defined as: Mw=xcexa3ni.Mi2 /xcexa3ni.Mi and Mp means the molecular mass of the quantitatively major species.
In the rest of the description, the molecular mass is expressed in polystyrene equivalents (Ep).
This preparative method is therefore not suitable if it is desired to prepare methylidene malonate nanoparticles constituted of:
polymers of average molecular mass between about 5000 and 10000, notably about 8000,
polymers of average molecular mass greater than 20000, without forming aggregates.
The present invention therefore consists of the preparation of methylidene malonate nanoparticles having a diameter of less than 500 nm, in particular 100 to 500 nm. formed from homogeneous molecular species of wide-ranging masses (Mw between about 2000 and 80000). The principle consists in dissolving the monomer in a water-miscible aprotic organic phase but which, under the conditions of preparation of the nanoparticles, forms, with the aqueous polymerisation medium, a non-solvent mixture of the polymer formed.
xe2x80x9cAprotic organic phasexe2x80x9d or xe2x80x9caprotic organic solventxe2x80x9d, is understood as meaning an organic phase or a solvent without labile proton which is capable of initiating an anion.
The advantages of this preparative method according to the invention are numerous:
it enables a more homogeneous dispersion of the monomer in the polymerisation medium,
it makes use of non-chlorinated solvents which are easy to evaporate since they are volatile,
it prevents the formation of polymer aggregates,
it gives rise to high polymerisation yields,
it enables the constitution of polymers of homogeneous wide-ranging molecular mass (Mw about 2000 to 100000, notably about 2000 to 80000) in forming nanoparticles having a diameter of less than 500 nm.
Furthermore, the method enables the use of dispersing agents such as non-ionic surfactants or colloid protecting polymers, which leads to particles having flexible surface properties.
Finally, the molecular mass of the oligomers/polymers which form the nanoparticles according to the invention can be perfectly mastered by adjusting the following preparative conditions:
the monomer concentration in the organic phase,
the pH and the molarity of the polymerisation medium,
the nature and the concentration of the dispersing agent,
the volume ratio of the aqueous phase (polymerisation medium)/organic phase,
the mode of introduction of the organic mixture in the aqueous phase.
In a 1st aspect therefore, the invention relates to a method for the preparation of nanoparticles formed from a random polymer of at least one compound of formula (I) 
in which
A represents a 
group or a 
group;
R1 and R2, identical or different, represent a linear or branched C1-C6 alkyl group;
n=1, 2, 3, 4 or 5;
characterised in that the monomer(s) is (are), before the polymerisation, dissolved in a water-miscible aprotic organic solvent forming, with the polymerisation medium, a non-solvent mixture of the polymer formed.
In an advantageous aspect, the invention relates to a method for the preparation of nanoparticles formed from a polymer of a compound of formula (I) 
in which
A represents a 
group or a 
group;
R1 and R2, identical or different, represent a linear or branched C1-C6 alkyl group;
n=1, 2, 3, 4 or 5;
characterised in that before the polymerisation, the monomer is dissolved in a water-miscible aprotic organic solvent forming, with the polymerisation medium, a non-solvent mixture of the polymer formed.
According to a particular aspect, the method according to the invention enables the preparation of nanoparticles having a diameter of less than 500 nm, preferably between 100 and 500 nm. and an average molecular mass (Mw) between about 1000 and 100000, notably between about 1000 and 80000. in particular between about 2000 and 80000, preferably between about 8000 and 80000.
In particular, the method according to the invention comprises the steps consisting in:
preparing a solution of at least one compound of formula (I) in a water-miscible aprotic organic solvent.
adding, with stirring, this organic phase to an aqueous polymerisation medium at a pH between 4.5 and 10,
recovering the nanoparticles thus obtained after homogenisation of the mixture and evaporating the organic solvent in vacuo.
The aqueous polymerisation medium can also be added to the organic phase which contains the monomer dissolved beforehand, and according to another aspect, the method according to the invention comprises the steps consisting in
preparing a solution of at least one compound of formula (I) in a water-miscible aprotic organic solvent,
adding, with stirring, to this organic phase, an aqueous polymerisation medium at a pH between 4.5 and 10,
recovering the nanoparticles thus obtained after homogenisation of the mixture and evaporating the organic solvent in vacuo.
As illustrated later on in the Examples, the pH of the polymerisation medium is selected as a function of the molecular mass of the polymer that is desired to prepare.
Advantageously, the mixture of the organic phase and the aqueous medium is homogenised by continuous stirring for about 30 minutes and then, optionally, the preparation is completed by distilled water.
The polymer formed precipitates in the polymerisation medium and can be recovered by filtration for example. The nanoparticle suspension thus obtained can then be conditioned and lyophilised.
The aprotic organic solvent used for dispersing the monomer(s) must be a solvent of said monomer(s) which should also be miscible with water. This solvent is preferably selected from acetone, acetonitrile, dioxane and tetrahydrofuran, acetone being particularly preferred.
Preferred aspects of the method are the following:
the concentration of monomer(s) of formula (I) in the organic solvent is of the order 30 mg/ml to 150 mg/ml;
the molarity of the polymerisation medium is of the order of {fraction (1/30)} M to ⅓ M;
volume ratio of the aqueous phase to the organic phase is between 3/1 and 20/1, preferably between 3/1 and 15/1.
Advantageously, the polymerisation medium contains one or more surfactants or colloid protectors.
The surfactants can be ionic or non-ionic surfactants for example. Non-ionic surfactants will preferably be used which are selected from copolymers of polyoxyethylene and polyoxypropylene, poloxamers and polysorbates. As colloid protector agents, polysaccharide derivatives will preferably be used, such as dextrans, hydrosoluble cellulose derivatives; polyethylene glycols; poly(vinyl alcohol).
Preferably, the compound polymerised to form the nanoparticles according to the method of the invention is a compound of formula (I) in which : A represents a 
group, n=1 and R1=R2=ethyl.
In another preferred aspect, the compound polymerised to form the nanoparticles according to the method of the invention is a compound of formula (I) in which: A represents a 
group, and R1=R2=propyl.
Advantageously, a mixture of compounds of formula (I) in which A is a 
group or a 
group as defined above, can also be random polymerised.
In a 2nd aspect, the invention relates to the nanoparticles formed from a random polymer of at least one methylidene malonate compound of formula (I), having a diameter of less than 500 nm, preferably between 100 and 500 nm and an average molecular mass (Mw) between about 1000 and 100000, notably between 1000 and 80000, in particular between about 2000 and 80000, preferably between about 8000 and 80000, obtainable by this method.
In particular, said nanoparticles, obtainable by this method, are formed from a polymer of a compound of formula (I), have a diameter of less than 500 nm, preferably between 100 and 500 nm and an Mw between about 1000 and 80000, in particular between about 2000 and 80000, preferably between about 8000 and 80000.
In a preferred aspect, the invention relates to nanoparticles formed from a random polymer of at least one compound of formula (I), having a diameter of less than 500 nm. preferably between 100 and 500 nm and an average molecular mass (Mw) between about 8000 and 100000, preferably between about 8000 and 80000.
In particular, the invention relates to nanoparticles formed from a polymer of a compound of formula (I), having a diameter of less than 500 nm, preferably between 100 and 500 nm and an average molecular mass (Mw) between about 8000 and 80000.
Advantageously, said nanoparticles are formed from a compound of formula (I) in which A represents a 
group, n=1 and R1=R2=ethyl.
In another preferred aspect, said nanoparticles are formed from a compound of formula (I) in which A represents a 
group and R1=R2=propyl.
Advantageously, said nanoparticles can be constituted of a random polymer of a mixture of compounds of formula (I) in which A is a 
group or a 
group as defined above.
According to a further aspect of the invention, said nanoparticles comprise, in their polymeric network, one or more biologically active molecules such as mentioned above.
In fact, in an advantageous aspect of the method according to the invention, the organic phase (when it is a biologically active molecule which is insoluble in water) or the polymerisation medium can contain one or more biologically active molecules.
xe2x80x9cBiologically active moleculexe2x80x9d is understood as meaning, in a non-limiting way, any molecule or macromolecule which has a prophylactic or curative biological activity, in vitro or in vivo, notably an anti-infectious agent, in particular an antiseptic agent, an antibiotic, an antiviral, an antiparasitic or antimitotic agent, notably an anticancer agent.
Antibiotic or antiseptic agents which can be used can be, for example, rifampicin and colistin.
As antiviral agents, didanosin, ribavirin, zidovudin, acyclovir, ganciclovir, foscarnet, vidarabin and zalcitabin can be cited in a non-limiting way.
Cis-plastin, 5-fluorouracil or taxol can, for example, be used as anti-cancer agents. Another advantageous antitumor agent is creatine phosphate whose activity is described in the application EP 0 614 366.
The invention also relates to pharmaceutical compositions containing said nanoparticles which comprise one or more biologically active molecules in association with a pharmaceutically acceptable vehicle.
The compositions according to the invention can be compositions which can be administered for example orally, sublingually, subcutaneously, intramuscularly, intravenously, transdermally, locally, rectally, via the pulmonary route, or nasally.
The suitable forms of administration notably comprise oral forms, such as tablets, gelatine capsules, powders, granules and oral solutions or suspensions, sublingual and buccal administration forms, as well as subcutaneous, intramuscular, intravenous, intranasal or intraocular and rectal administration forms.